The present invention relates to a method of perfusion culture using a naturally-occurring agglutinin.
It has been strongly desired to develop a technique for producing useful bioactive substances such as hormones, cytokines, and monoclonal antibodies in a large amount by culturing a large amount of cells. In consideration of an industrial scale production, suspension culture, in which cells are proliferated while floating in a culture medium without attaching to a culture immobilization carrier, is considered as the most useful method since the production is easily scaled up.
However, in the cell culture, since growth inhibitors are excreted from the cells, cell proliferation will stop at a relatively low cell density unless the culture medium containing the growth inhibitor is removed. Hence, to culture cells in a large amount and with a high density, it is necessary to perform perfusion culture while old culture medium containing the growth inhibitors is appropriately discharged from a vessel and replaced with fresh culture medium.
To perform the perfusion culture, it is very important to precipitate living cells quickly in the culture medium in order to separate the living cells and the old culture medium, thereby discharging the old culture medium from a culture vessel.
Conventional methods to separate the culture medium and the living cells include (1) a method using a filter, (2) a method using gravity, (3) a method using a centrifuge, and (4) a method immobilizing cells to a carrier.
However, the method (1) has a problem in that filter clogging takes place. The method (2) has a problem in that separation efficiency is low since the specific gravity of animal cells is about 1.1. The method (3) has problems in that the apparatus has a complicated structure, and that the centrifugal force will have an adverse effect on the cells. The method (4) has drawbacks in that a troublesome operation is required for immobilization, and that it is difficult to scale up the production. As described, none of them is a satisfactory method for culturing a large amount of cells.
Furthermore, Japanese Patent Application KOKAI Publication No. 1-165374 discloses a method for improving separation efficiency of the cells from a culture medium by agglutinating the cells by addition of a mixture of polyacrylic acid and chitosan as an agglutinant, to the culture medium. All drawbacks associated with the aforementioned methods can be overcome by this method.
However, this method has the following disadvantages.
(i) Polyacrylic acid and chitosan, which are non-biological substances, may possibly cause cytotoxicity.
(ii) Relatively large amount of polyacrylic acid/chitosan (50-200 .mu.g/mL) is needed to effectively agglutinate animal cells.
(iii) Chitosan produced from chitin by its heat treatment under strong alkali condition is not uniform from lot to lot. This non-uniformity is inappropriate for the process of producing medicine with respect to safety.
(iv) Agglutinate formed with polyacrylic acid/chitosan are too large (0.1 to 2.0 mm in diameter) for its core to be supplied with enough oxygen and nutrients.